In vitro culturing of cells provides material necessary for research in pharmacology, physiology, and toxicology. Recent advances in pharmaceutical screening techniques allow pharmaceutical companies to rapidly screen vast libraries of compounds against therapeutic targets. These large-scale screening techniques require large numbers of cells grown and maintained in vitro. Growing and maintaining these large numbers of cells requires large numbers of cells as well as large volumes of cell growth media and reagents and large numbers and types of laboratory cell culture containers and laboratory equipment. This activity is expensive and labor intensive. Large-scale screening techniques provide market pressure for the development of new and improved cell culture techniques to provide more efficient and less expensive large-scale cell culture equipment.
In addition to the challenges of large-scale culture, particular cell types can represent special problems. For example, growing stem cells in culture can be quite difficult. A successful stem cell culture provides conditions that keep stem cells either dividing but not differentiating or differentiating into a defined cell type. It is desirable to be able to maintain stem cells in an undifferentiated state, and to control the transition of stem cells in culture from an undifferentiated to a differentiated state. These cells may not be robust in culture, and may require very specific cell culture conditions in order to thrive. Each of these conditions represent significant challenges for cell culture, especially large-scale cell culture.
Currently, many researchers find that successful conditions for stem cell growth require the presence of a layer of non-stem cells in the same culture environment as the stem cells. These non-stem cells are called “feeder cells” and are often grown as a first layer of cells in a stem cell culture. These feeder cells are often mouse cancer cells, but they may also be derived from human or other animal sources. The feeder layer provides a source of biologically active components that nourish the stem cells. They may also provide an extracellular environment for the stem cells that allows the stem cells to adhere to a surface in culture. The feeder cells may be irradiated to prevent them from growing. However, it is often difficult to separate stem cells from the feeder cells that are co-cultured in a single cell culture vessel. The process of purifying one cell type from the other is fallible, and the stem cell culture can become contaminated with feeder layer cells. It is desirable to provide devices and methods for growing multiple cell types together in culture, for example to allow feeder cells to share biologically active components with stem cells (“paracellular communication”), but yet allow for the cell types to be reliably separated from each other.
Feeder cells can be used to “condition” cell culture media. For example, feeder cells can be grown in media for a time, and that conditioned media can be harvested and used, processed or unprocessed, as growth media for stem cells. This step of conditioning cell culture media is time consuming and introduces added risk of contamination by introducing additional handling steps.
In addition, oxygen tension is a cell culture parameter that has been found to have a regulatory function in the differentiation of stem cells. For example, stem cells may grow in an undifferentiated state better in a relatively low oxygen environment, while feeder cells may prefer a relatively high oxygen environment. A vessel that permits better control of cell culture parameters such as oxygen content for co-cultured cells would also be beneficial.
In addition, cell-based high throughput applications have become automated. Automation permits manipulation of the cell culture vessel, such as roller bottles, cell culture dishes and plates, multiwell plates, microtiter plates, common flasks and multi-layered cell growth flasks and vessels, much like the manipulations performed by the manual operator. It is desirable to provide cell culture vessels which are amenable to automation systems that are currently available and to automation systems that are in development. Additionally, it is desirable that the cell culture apparatus will be suitable for use in the performance of high throughput assay applications that commonly employ robotic manipulation.
Further, flask vessels having multiple layers of cell growth are capable of producing a greater cell yield than commonly known flasks that permit growth of cells on a single bottom wall. There is a need for multi-layered cell culture flasks or vessels that provide for the co-culture of cells and the effective separation of co-cultured cells one from the other, while still providing adequate gas exchange.